Dissertation
zur Erlangung des Doktorgrades
der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August-Universität
vorgelegt von Olav Grundmann
aus Göttingen
Göttingen 2001
Korreferent: Prof. Dr. F. Mayer
Tag der mündlichen Prüfung: 27.Juni.2001
Table of Contents
Summary
... 1Zusammenfassung
... 3Chapter I: Introduction
... 5Regulation of morphology of the bakers’ yeast Saccharomyces cerevisiae in dependence on the nutritional conditions... 5
Life cycle of Saccharomyces cerevisiae... 5
Pseudohyphal growth of Saccharomyces cerevisiae... 6
Environmental stimuli and sensing systems... 7
Signaling pathways... 8
FLO11 is indispensable for filamentous growth... 11
Why studying pseudohyphal development in Saccharomyces cerevisiae?.... 12
Regulation of amino acid biosynthesis in the yeast Saccharomyces cerevisiae... 13
General control of amino acid biosynthesis... 13
General structure of DNA binding transcription factors... 14
Gcn4p is the final effector of the general control in Saccharomyces cerevisiae... 15
Initiation of translation and its regulation in eukaryotes... 16
Translational control of GCN4 expression... 19
Aim of this work... 23
References... 24
Chapter II:
Repression of GCN4 mRNA translation by nitrogen starvation in
S. cerevisiae
... 31Summary... 31
Introduction... 32
Materials & Methods... 34
Results... 40
Discussion... 58
References... 61
Chapter III: Amino acid starvation and Gcn4p activate adhesion and FLO11 gene expression in yeast
... 65Summary... 65
Introduction... 66
Materials & Methods... 68
Results... 72
Discussion... 88
References... 92
Chapter IV:
Genome-wide transcription profile analysis of Gcn4p-dependent gene transcription under amino acid starvation conditions in
Saccharomyces cerevisiae
... 96Summary... 96
Introduction... 97
Materials & Methods... 99
Results... 101
Discussion... 106
References... 141
Lebenslauf
... 145Summary
Under amino acid starvation conditions, the bakers’ yeast Saccharomyces cerevisiae activates a system called “General control of amino acid biosynthesis”. Gcn4p, the transcription factor of this system induces the expression of more than 50 genes involved in the different amino acid biosynthetic pathways. In this thesis it could be shown that during simultaneous limitation of amino acids and nitrogen the general control is not activated. More exactly, even a decrease of the Gcn4p activity was detected, which was traced back onto a reduction of the Gcn4 protein amount in the cell.
This decrease of the intracellular concentration was caused by translational control of the GCN4 mRNA, which was able to repress even a 2-fold increase of the GCN4 transcription rate. Furthermore during nitrogen starvation conditions no correlation between the stature of eIF-2 phosphorylation and GCN4 expression was observed. For this reason an involvement of the already known mechanism of translational regulation of GCN4 mRNA could be excluded. Rather a factor is postulated, which is situated downstream of eIF-2 and has a regulatory effect on initiation of translation.
Although it could be proven that the Gcn4p activity is repressed during amino acid starvation, a gcn4D mutant strain was not able to form pseudohyphae any more. This indicated to a dependence of pseudohyphal growth on the presence of Gcn4p.
Furthermore it was detected that the transcription of FLO11, which is a flocculin gene necessary for pseudohyphal growth, was activated by amino acid limitation under normal nitrogen concentrations as well. This activation is Gcn4p-dependent and leads to an improved cell-cell adhesion. Gcn4p as well as the transcription factor of the cAMP pathway, Flo8p are necessary for FLO11 expression during amino acid limitation. On the other hand the transcription factors of the MAPK pathway, Ste12p and Tec1p, which are important under nitrogen starvation conditions as well have only a minor importance.
By comparative transcriptome analysis of a yeast wild type and a gcn4D mutant strain, which were grown under amino acid limitation conditions, 225 genes were identified, which were Gcn4p-dependent activated during amino acid starvation conditions. Not only genes of amino acid or nucleotide metabolism, for which a Gcn4p-dependent transcription was already known, but also several other genes were identified, which are involved in completely different cellular processes. A
Gcn4p-dependent activation of transcription could be detected for genes of carbon, fatty acid and phosphorus metabolism, as well as for genes coding for chromatin structure determining proteins (e.g. histones).
Zusammenfassung
Unter Aminosäure-Mangelbedingungen schaltet die Bäckerhefe Saccharomyces cerevisiae die sogenannte „Allgemeine Kontrolle der Aminosäure-Biosynthese“ ein. Gcn4p, der Transkriptionsfaktor dieses Systems, aktiviert die Expression von über 50 Genen, die in die verschiedenen Aminosäure-Biosynthesewege involviert sind. Im Rahmen dieser Arbeit konnte gezeigt werden, daß bei gleichzeitiger Limitierung von Aminosäuren und Stickstoff, die Allgemeine Kontrolle der Aminosäure-Biosynthese nicht aktiviert wird. Vielmehr konnte eine Abnahme der Gcn4p-Aktivität beobachtet werden, die auf eine Verminderung der Gcn4-Proteinmenge zurückzuführen war. Ursache für diese Abnahme der intrazellulären Proteinkonzentration war die translationelle Kontrolle der GCN4-mRNA, die selbst eine unter diesen Bedingungen auftretende zweifache Erhöhung der GCN4-Transkriptionsrate repremieren konnte. Außerdem wurde gezeigt, daß unter Stickstoff-Mangelbedingungen keine Korrelation zwischen dem Phosphorylierungsstatus von eIF-2 und der GCN4-Expression mehr gegeben war. Dadurch konnte ausgeschlossen werden, daß der bereits bekannte Mechanismus der translationellen Kontrolle der GCN4-mRNA unter diesen Bedingungen eine Rolle spielt. Vielmehr wird ein zusätzlicher Faktor postuliert, der unterhalb von eIF-2 direkt auf die Translationsinitiation wirkt.
Obwohl nachgewiesen werden konnte, daß die Gcn4p-Aktivität unter Stickstoff-Mangelbedingungen repremiert wird, war eine gcn4D-Mutante nicht mehr in der Lage, Pseudohyphen zu bilden. Dies zeigt eindeutig, daß eine Abhängigkeit des Pseudohyphen-Wachstums von der Anwesenheit von Gcn4p gegeben ist. Darüber hinaus wurde festgestellt, daß die Transkription des für das Pseudohyphen-Wachstum notwendigen Flocculin-Gens, FLO11 auch durch Aminosäure-Mangel unter normalen Stickstoff-Konzentrationen aktiviert werden kann. Diese Aktivierung ist Gcn4p-abhängig und führt zu einer verstärkten Zell-Zell Adhäsion. Neben Gcn4p ist für die FLO11-Expression unter Aminosäure-Mangelbedingungen auch der Transkriptionsfaktor des cAMP-Weges, Flo8p notwendig. Hingegen spielen die für die FLO11-Expression unter Stickstoff-Mangelbedingungen gleichfalls wichtigen Transkriptionsfaktoren des MAPK-Weges, Ste12p und Tec1p, eine eher untergeordnete Rolle.
Durch vergleichende Transkriptom-Analyse eines Hefe-Wildtyp-Stammes und eines gcn4D-Deletionsstammes, die unter Aminosäure-Mangelbedingungen gewachsen waren, konnten 225 Gene identifiziert werden, die unter Mangelbedingungen Gcn4p-abhängig induziert werden. Neben Genen des Aminosäure-oder Nukleotid-Metabolismus, für die eine Gcn4p-abhängige Transkription bereits bekannt war, konnten auch eine Vielzahl von Genen identifiziert werden, die in völlig andere zelluläre Prozesse involviert sind. So wurde eine Gcn4p-abhängige Transkriptionsaktivierung für Gene des Kohlenstoff-, Fettsäure- und Phosphat-Stoffwechsels, aber auch für Gene, die für Chromatinstruktur determinierende Proteine codieren (z.B. Histone), nachgewiesen.
Chapter I
Introduction
Regulation of morphology of the bakers’ yeast Saccharomyces cerevisiae in dependence on the nutritional conditions.
Life cycle of Saccharomyces cerevisiae.
The budding yeast S. cerevisiae is able to change its morphology and interconvert between a unicellular and a multicellular filamentous growth type. Furthermore, the organism can grow in a haploid or a diploid form (Figure 1). In the laboratory, yeast strains are usually cultivated on media containing a fermentable carbon source and sufficient nitrogen, such as glucose and ammonium, respectively. On these media haploid cells are sticky and smaller than diploid cells, but their overall morphology is similar. They show a unicellular ellipsoid cell morphology, called yeast form (YF).
During growth of diploids, depletion of both nutrients leads to sporulation (meiosis), whereas limitation of only one nutrient source either favors growth arrest (carbon starvation) or induces filamentous growth (nitrogen starvation) as chains of elongated cells, called pseudohyphae (PH). During prolonged growth of haploids on complex media with glucose limitation, the so-called haploid invasive growth is observed, characterized by the development of small microfilaments, which stick on the surface of the agar plate (Cullen & Sprague, 2000).
Additionally, both cellular forms can be distinguished by their budding pattern (Mösch, 2000): diploid yeast form cells exhibit a bipolar budding pattern, which switches to a unipolar distal mode during pseudohyphal growth. This budding at the opposite pole of the birth end is essential for establishment of a filamentous growth mode. Haploid cells always bud unipolar at the proximal pole, that means, budding is constrained to the mother-daughter neck region.
Figure 1. Life cycle of the bakers’ yeast Saccharomyces cerevisiae.
Haploid as well as diploid cells can adopt yeast form vegetative growth, invasive growth or stationary phase arrest. Upon nitrogen limitation, diploid cells switch their morphology and grow invasively as multicellular filaments called pseudohyphae.
Haploid invasive growth occurs during prolonged growth on complex media with glucose limitation leading to the formation of small microfilaments. Haploid cells (1n) conjugate to form diploids (2n) and these can sporulate to form haploids. General nutrient limitation results in stationary phase arrest in the haploid as well as in the diploid form.
Pseudohyphal growth of Saccharomyces cerevisiae.
As described before, the development of pseudohyphae in S. cerevisiae demands at least two stimuli, starvation for nitrogen and the presence of a fermentable carbon source. The switch from the yeast form to pseudohyphal growth is accompanied by alterations in several distinct cellular processes: (i) Cell morphology changes from ellipsoidal shaped yeast form cells to long, thin pseudohyphal cells. (ii) The budding pattern of the cells is altered from bipolar to unipolar distal, resulting in linear filamentous chains of cells. (iii) Furthermore, cell separation switches from complete to incomplete division, which means that cells remain attached to each other and form long multicellular chains. (iv) The process of cell division is modified during pseudohyphal development as well. All pseudohyphal growth occurs during the budded period (G2-phase) of the cell cycle, so that mitosis is restricted until the bud has reached the cell
Conjug ation
size of the mother cell. Consequently, both cells bud synchronously in the following cycle without G1-delay (Mösch, 2000).
Taken together, these changes enable pseudohyphae to grow invasive, in contrast to superficial growth of yeast form cells (Figure 2).
Figure 2. Comparison of the growth behavior of yeast form and pseudohyphal cells of S. cerevisiae (according to Mösch, 2000).
Diploid S. cerevisiae cells were streaked on either rich medium or nitrogen starvation medium to obtain single colonies. Microcolonies of cells growing as yeast form (YF) or as pseudohyphae (PH) were photographed after 17 h of incubation at 30°C.
Environmental stimuli and sensing systems.
However, yeast cells must be able to sense both, abundant fermentable carbon source as well as nitrogen deprivation to undergo pseudohyphal development. Easy utilizable nitrogen sources like ammonium or arginine suppress pseudohyphal formation in standard concentrations, whereas standard amounts of proline or uracil are permissive for the formation of pseudohyphae (Gimeno et al., 1992). The sensor systems that differentiate between diverse nitrogen compounds and control pseudohyphal growth are largely unknown. For sensing ammonium availability the membrane-bound high-affinity ammonium permease Mep2p is already described (Lorenz & Heitman, 1998).
Strains with deletions in the MEP2 gene are unable to form pseudohyphae under ammonium starvation conditions, suggesting that Mep2p, besides its function in ammonium uptake, additionally transduces the signal to intracellular signaling pathways.
Yeast form (YF)
Rich medium
Pseudohyphae (PH)
N-starvation medium
Besides nitrogen, carbon is the other nutrient crucial for pseudohyphal differentiation. It has to be fermentable like glucose, galactose or raffinose and should be available in surplus to prevent cells from sporulation. Membrane-bound or membrane-associated sensors regulating pseudohyphal development in response to the presence of fermentable carbon sources are also unknown up to now. Nevertheless, it has been reported that components of the Ras/cAMP pathway are involved in the perception of extracellular glucose concentrations (Broach, 1991). Under conditions of nitrogen starvation, activation of the small GTP-binding protein Ras2p indeed induces hyperfilamentous growth. This indicates that Ras2p may be a transmitter that regulates pseudohyphal development in response to glucose availability. A possible sensor for the Ras2p dependent glucose signal could be the membrane-bound Gpr1p/Gpa2p complex (Lorenz et al., 2000). Recently, a suppression of pseudohyphal development was detected in GPR1 deletion strains grown on glucose, whereas on media containing maltose this suppression was not observed, implicating an involvement of Gpr1p in glucose perception (Lorenz et al., 2000). The influence of other stimuli, such as osmolarity, pH or warmth on pseudohyphal development of S. cerevisiae has not yet been investigated, whereas these environmental conditions are known to be important for dimorphism of pathogenic fungi like Candida albicans (Soll, 1997).
Haploid strains also change their growth phenotype depending on environmental stimuli. Contrary to diploid strains, it is believed that this morphological change occurs under non-starvation conditions (Banuett, 1998; Madhani & Fink, 1998). Most recent studies demonstrated that haploid invasive growth is also induced by starvation conditions suggesting glucose limitation as stimulus for the invasive growth behavior (Cullen & Sprague, 2000; Madhani, 2000). This is supported by the observation that invasive growth on agar plates containing 2 % glucose does not appear until a few days of growth, when the glucose concentration has been reduced by consumption that limitation conditions are evident.
Signaling pathways.
The regulation of pseudohyphal development is a complex process, involving at least two separate, but interconnected signaling pathways (Figure 3) (Pan et al., 2000; Rupp et al., 1999). One is the cAMP pathway, which was identified in a yeast strain showing a hyperfilamentous growth phenotype due to a dominant activated Ras2 protein
(Ras2Val19p) (Gimeno et al., 1992). This small GTP-binding protein is known to elevate the intracellular cAMP levels by stimulating the adenylyl cyclase Cyr1p. High levels of cAMP in turn remove the inhibitory subunit Bcy1p, from one of the three catalytic subunits of proteinkinase A, Tpk1p, Tpk2p and Tpk3p, respectively (Broach, 1991).
Although for pseudohyphal differentiation only Tpk2p is necessary, all three subunits are redundant for viability (Robertson & Fink, 1998). One of the Tpk2p target proteins is the transcriptional repressor Sfl1p, which negatively regulates transcription of FLO11, encoding a cell surface flocculin that is strictly required for flocculation and pseudohyphal growth (Lo & Dranginis, 1998). An additional target of Tpk2p is Flo8p, a transcription factor acting positively on FLO11 transcription (Rupp et al., 1999).
Mutants in the FLO8 gene have been shown to be unable to form pseudohyphae.
Nevertheless, not all genetic backgrounds of S. cerevisiae are able to form pseudohyphae, e.g. S288C strains are naturally defective in pseudohyphal development, due to a mutation in the FLO8 gene that leads to the formation of an unfunctional polypeptide (Liu et al., 1996). Conclusively, S288C strains transformed with an intact FLO8 gene regain the ability for pseudohyphal growth, suggesting a quite important role of Flo8p in pseudohyphal differentiation. Another activator of the cAMP pathway is the Gpr1/Gpa2 protein complex, which possibly senses glucose availability (see before) (Kübler et al., 1997; Lorenz & Heitman, 1997; Lorenz et al., 2000).
The other signal transduction pathway known to be involved in regulating pseudohyphal growth is the MAPK (mitogen-activated protein kinase) cascade that is also important for signaling during the mating process of haploid S. cerevisiae cells.
Four proteins of this MAPK cascade are functional in the pheromone response as well as in pseudohyphal formation, the p65PAK kinase homolog Ste20p, Ste11p (MAPKKK), Ste7p (MAPKK), and the transcription factor Ste12p (Liu et al., 1993). For signaling during filamentous growth, the MAPK of the pheromone response Fus3p is replaced by Kss1p (Cook et al., 1997; Madhani et al., 1997). Upon non-induced conditions, unphosphorylated Kss1p inhibits the transcription factor Ste12p via Dig1p and Dig2p, and thus prevents the activation of Ste12p-dependent pseudohyphal development (Cook et al., 1996). During activation of the MAPK cascade, Kss1p is phosphorylated and stimulates Ste12p, which promotes transcription of the target genes. Nevertheless, Ste12p requires an additional transcription factor for the activation of pseudohyphae inducing genes (Gavrias et al., 1996). This transcription factor is Tec1p. Ste12p and
Tec1p are able to bind as a heterodimer to specific cis sequences termed filamentation responsive elements (FREs) (Madhani & Fink, 1997). FREs are not only necessary but also sufficient to direct pseudohyphal specific gene expression. They are present in the promoter regions of at least two genes required for pseudohyphal development, TEC1 and FLO11.
Upstream components that activate the MAPK cascade during pseudohyphal differentiation comprise the small GTP-binding proteins Ras2p and Cdc42p (Mösch &
Fink, 1997; Mösch et al., 1996; Roberts et al., 1997). In addition to these components, the two yeast homologs of 14-3-3 proteins, Bmh1p and Bmh2p, are implicated in the filamentation MAPK cascade. These proteins appear to regulate transcriptional induction and pseudohyphal cell elongation independently of each other, and their action may be exerted by interactions with the Ste20 protein (Roberts et al., 1997).
Interestingly, under conditions of nitrogen limitation Ras2p is involved in both pathways, the cAMP pathway as well as the MAPK cascade. A variety of other proteins are also involved in the regulation of pseudohyphal differentiation such as Phd1p and Sok2p (Gimeno & Fink, 1994; Ward et al., 1995). Genetic studies indicated that Phd1p activates pseudohyphal growth, whereas Sok2p seems to be an antagonist of Phd1p. Up to now it is not known, if Phd1p and Sok2p act in a linear pathway or operate on the same target.
Furthermore, proteins important for cell morphology like Bud8p and Bud9p are involved in pseudohyphal development (Taheri et al., 2000). These membrane-bound proteins determine cell polarity and therefore the budding pattern of the cell. But the target sequences of Bud8p and Bud9p are still unknown.
Figure 3. Model of signaling pathways regulating pseudohyphal development in S. cerevisiae .
See text for details.
FLO11 is indispensable for filamentous growth.
The S. cerevisiae genome contains a family of cell-wall proteins related to the adhesins of pathogenic fungi. One branch of this protein family, encoded by genes including FLO1, FLO5, FLO9 and FLO10, is called the ‘flocculins’ (Caro et al., 1997), as these proteins promote cell-cell adhesion to form multicellular clumps that sediment out of solution (Teunissen & Steensma, 1995). The FLO1, FLO5, FLO9 and FLO10 genes share considerable sequence homology. A second group of Flo family members
Cdc42p Ras2p
has a domain structure similar to that of the first group, but with quite distinct primary sequences. This second group includes three proteins, Flo11p, Fig2p and Aga1p (Guo et al., 2000). Fig2p and Aga1p are induced during mating (Erdman et al., 1998), whereas Flo11p is required for diploid pseudohyphal formation and haploid invasive growth (Lo
& Dranginis, 1998; Roberts & Fink, 1994). FLO11 expression is controlled by the cAMP as well as the MAPK pathway, which supports the importance of regulating FLO11 expression in response to different environmental signals (Rupp et al., 1999).
Strains impaired in pseudohyphae formation, like tpk2D, flo8D, ste12D or tec1D null mutants, exhibit decreased FLO11 expression levels, too.
Contrarily, deletion of inhibitors of pseudohyphal formation, like Sfl1p, results in an activation of FLO11 expression (Robertson & Fink, 1998). Other transcription factors necessary for the regulation of pseudohyphal development also bind in the FLO11 promoter region such as Phd1p. To make all these interactions possible, that means to integrate all these signals, the FLO11 promoter is extraordinary large comprising up to 3000 kb, thus making it to the largest promoter known in yeast (Rupp et al., 1999).
Taken together, FLO11 displays an excellent reporter gene for studying the influence of nutrients and other environmental stimuli on pseudohyphae formation. Nevertheless, the whole process of pseudohyphal development is much more complex involving the activation of several other genes. For instance, the MAPK pathway is additionally required for cell elongation, whereas the cAMP pathway modulates the transition from bipolar to unipolar budding, too.
Why studying pseudohyphal development in Saccharomyces cerevisiae?
S. cerevisiae is a model organism for studying regulatory mechanisms, because cells are able to grow in a haploid as well as a diploid form so that genetic studies are relative simple to carry out. Furthermore, the genome of the yeast strain S288C is sequenced completely and many protocols and tools for the manipulation of the organism are available, forming a good base for investigations. Additionally, many proteins and signal transduction pathways are conserved throughout yeast and higher eukaryotes, which are not so easy to handle. So, results obtained from experiments in yeast may help elucidating and understanding the function of proteins and complex pathways in higher eukaryotes much faster.
One example for the elucidation of a complex regulatory system is the pseudohyphal differentiation of the bakers’ yeast S. cerevisiae, which has features in common with dimorphism of pathogenic fungi, in which dimorphic transition is often correlated with pathogenicity (Soll, 1997). The molecular models drawn from pseudohyphal growth in S. cerevisiae have turned out to be true for human and plant pathogens as well.
Homologs of the G-proteins, the protein kinases and the transcription factors required for pseudohyphal signaling in S. cerevisiae have been found to control hyphal
Homologs of the G-proteins, the protein kinases and the transcription factors required for pseudohyphal signaling in S. cerevisiae have been found to control hyphal